PROJECT 3 CONSEQUENCE OF NOVEL COCAINE PHARMACOTHERAPIES ON NEUROCHEMISTRY IN RODENT MODELS OF COCAINE DEPENDENCE Allyn Howlett, Director; Rong Chen and Steven Childers, Co-Investigators SUMMARY Cocaine addiction produces neuroadaptations in multiple neurotransmitter systems that perpetuate cocaine relapse. We hypothesize that neurochemical neuroadaptions resulting from cocaine self-administration will be modified by drug combinations that consists of an agonist pharmacotherapy with a cognitive remediator. Project 3 will delineate how these neuroadaptions can be treated with a cocaine-substitute (agonist) drug, phendimetrazine (active form is phenmetrazine (PM)), combined with Drug B candidates that serve as cognitive remediators, as identified by cognition testing in Project 1. We will examine the cellular and molecular mechanisms by which these combination medications modify brain neurochemistry to reduce cocaine self- administration and reverse cognitive deficits produced by cocaine. Aim 1 will determine the impact of PM in combination with Drug B cognitive remediators on neuroadaptations associated with chronic cocaine self- administration in rats, beginning with the mGluR2/3 agonist LY-379268. We will examine the ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex (PFC) and other limbic areas for changes in: 1) gene expression determined by quantitative PCR; 2) membrane expression of receptors and transporters assessed by radioligand binding; 3) receptor function by GTP?S binding; 4) trafficking of receptors and transporters assayed by tissue fractionation and Western blotting; 5) signal transduction determined by phosphorylation of regulatory proteins. Aim 2 will determine the differential role of D2 autoreceptors and postsynaptic D2 receptors in NAc (using Intracranial lentivirus delivering siRNA constructs) in the mechanism of action of drug combination pharmacotherapies determined by Project 1. Experiments will determine: neuroadaptations of intra-VTA or intra-NAc knockdown of D2 receptors; how neuroadaptations of PM alone or in combination with drug B are modified by D2 autoreceptor or postsynaptic D2 receptor knockdown; how neuroadaptations of cocaine self-administration are modified by D2 autoreceptor or postsynaptic D2 receptor knockdown; and the efficacy of drug combinations on normalization of neuroadaptations in D2 knockdown animals self- administering cocaine. These neurochemical data will explain mechanisms for the development of addiction and cognitive deficits associated with cocaine self-administration, and will define how PM and cognition remediators function at the neuronal level. Since the animals used in Project 3 will be essentially the same as those used in Project 2, biochemical data from Project 3 can be directly compared with voltammetry, microdialysis, and neuronal activity data from Project 2. These results will provide pharmacodynamic evidence needed for rational design of clinical studies of novel drug combinations in addiction treatment.